A variety of cell-based assays are of considerable commercial relevance in screening for modulators of cell-based activity. For example, compounds which affect cell death can have profound biological activities and are desirably screened for in cell-based assays. Cell death has become recognized as a physiological process important in normal development, hormonal regulation of various tissues, and, e.g., in regulation of the receptor repertoires of both T and B lymphocytes. The finding that a pattern of morphological changes is common to many examples of programmed cell death (or PCD) led to the suggestion of a common mechanism, and the term xe2x80x9capoptosisxe2x80x9d was defined to include both the morphological features and the mechanism common to such programmed cell death (Kerr et al., Br. J. Cancer 26:239). This concept was extended by the finding that nuclear DNA fragmentation correlates well with apoptotic morphology (Arends et al., Am. J. Pathol. 136:593 (1990)), and the scientific literature contains many examples of PCD accompanied by these features. There are also clear examples of PCD in the absence of apoptotic morphology or DNA fragmentation (Clarke, Anat. Embryl. 181:195 (1990), Martin et al, J. Cell Biol. 106:829 (1988), and Ishigami et al., J. Immunol. 148:360 (1992)).
Cell-based assay systems model relevant biological phenomena, and have generally been widely adopted as screening assays, e.g., when screening for a compound""s effect(s) on apoptosis or other biological phenomena. Pioneering technology providing cell- and other particle-based microscale assays are set forth in Parce et al. xe2x80x9cHigh Throughput Screening Assay Systems in Microscale Fluidic Devicesxe2x80x9d WO 98/00231; in PCT/US00/04522, filed Feb. 22, 2000, entitled xe2x80x9cManipulation of Microparticles In Microfluidic Systems,xe2x80x9d by Mehta et al.; and in PCT/US00/04486, filed Feb. 22, 2000, entitled xe2x80x9cDevices and Systems for Sequencing by Synthesis,xe2x80x9d by Mehta et al.
Other cell-based assays include various methods for the preparative or analytic sorting of different types of cells. For example, cell panning generally involves attaching an appropriate antibody or other cell-specific reagent to a solid support and then exposing the solid support to a heterogeneous cell sample. Cells possessing, e.g., the corresponding membrane-bound antigen will bind to the support, leaving those lacking the appropriate antigenic determinant to be washed away. Other well-known sorting methods include those using fluorescence-activated cell sorters (xe2x80x9cFACSsxe2x80x9d). FACSs for use in sorting cells and certain subcellular components such as molecules of DNA have been proposed in, e.g., Fu, A. Y. et al. (1999) xe2x80x9cA Microfabricated Fluorescence-Activated Cell Sorter,xe2x80x9d Nat. Biotechnol. 17:1109-1111; Unger, M., et al. (1999) xe2x80x9cSingle Molecule Fluorescence Observed with Mercury Lamp Illumination,xe2x80x9d Biotechniques 27:1008-1013; and Chou, H. P. et al. (1999) xe2x80x9cA Microfabricated Device for Sizing and Sorting DNA Molecules,xe2x80x9d Proc. Nat""l. Acad. Sci. 96:11-13. These sorting techniques utilizing generally involve focusing cells or other particles by flow channel geometry.
While cell-based assays are generally preferred in certain microscale screening applications, certain of these assays are difficult to adapt to conventional notions of high-throughput or ultra high-throughput screening assay systems. For example, one difficulty in flowing assay systems is that, during pressure-based flow of fluids in channels, non-uniform flow velocities are experienced. Faster fluid and material flow is observed in the center of a moving fluid stream than on the edge of a moving fluid stream. This non-uniform flow velocity reduces throughput for flowing assays, because assay runs have to be spaced well apart in the fluid stream to prevent overlap of materials moving at different velocities.
Accordingly, it would be advantageous to provide mechanisms for facilitating cell-based assays, including cell sorting techniques, especially in microscale systems. Additional microscale assays directed at subcellular-components, such as nucleic acids would also be desirable. The present invention provides these and other features which will become clear upon consideration of the following.
The present invention relates to methods of focusing particles in microchannels, e.g., to improve assay throughput and sensitivity, to sort particles, to count particles, or the like. In the methods of the invention, cells and other particles are focused in the center of, to one side of, or in other selected regions of microscale channels, thereby avoiding, e.g., the above noted difficulties inherent in pressure-based flow of particles. Furthermore, the device structures of the present invention are optionally integrated with other microfluidic systems. Other reactions or manipulations, including various binding assays, involving cells, other particles, or fluids upstream of the detection zone are also optionally performed, e.g., monitoring drug interactions with cells or other particles.
In one aspect, the invention provides methods of providing substantially uniform flow velocity to particles flowing in a first microchannel. In the methods, the particles are optionally flowed in the microchannel, e.g., using pressure-based flow, in which the particles flow with a substantially non-uniform flow velocity. Prior to performing the flowing step, the particles are optionally sampled with at least one capillary element, e.g., by dipping the capillary element into a well containing the particles on a microwell plate and drawing the particles into, e.g., reservoirs, microchannels, or other chambers of the device. The particles (e.g., a cell, a set of cells, a microbead, a set of microbeads, a functionalized microbead, a set of functionalized microbeads, a molecule, a set of molecules, etc.) are optionally focused horizontally and/or vertically in the first microchannel to provide substantially uniform flow velocity to the particles in the first microchannel. Particles are optionally focused using one or more fluid direction components (e.g., a fluid pressure force modulator, an electrokinetic force modulator, a capillary force modulator, a fluid wicking element, or the like). Additional options include sorting, detecting or otherwise manipulating the focused particles. Advantages of the invention include that the substantially uniform flow velocity increases a rate of particle mixing which reduces dispersion of the particles flowing in the first microchannel. For example, the particles are typically focused proximal to a center portion of the first microchannel, e.g., where focused fluid streamlines, that include the particles, flow at higher flow velocities than fluid streamlines flowing proximal to a surface of the first microchannel.
The particles are horizontally focused in the microchannel, e.g., by introducing a low density fluid and a high density fluid into the microchannel, causing the particles to be focused in an intermediate density fluid present between the high density fluid and the low density fluid. The particles are also optionally focused in a top or a bottom portion of the microchannel by introducing a high or a low density fluid into the microchannel with the flowing particles. The particles are vertically or horizontally focused in the microchannel, e.g., by simultaneously introducing fluid flow from two opposing microchannels into the first microchannel during flow of the particles in the first channel. Vertical focusing is also optionally achieved to one side of a microchannel by simultaneously introducing fluid flow from, e.g., a second microchannel into the first microchannel during flow of the particles in the first microchannel.
In another aspect, the particles include a plurality of first and second components in which the first components focus more than the second components such that at least some of the first components separate from the second components in the first microchannel. In preferred embodiments, substantially all of the first components separate from the second components in the first microchannel. In addition, the first components generally focus proximal to a center portion of the first microchannel more than the second components due to greater dispersion of the second components relative to the first components. The center portion of the first microchannel typically includes a higher velocity flow stream than other flow streams in the first microchannel, which effects the separation of first and second components.
Each first component optionally includes, e.g., a cell, a bead (e.g., a microbead, etc.), or the like, and typically further includes at least one antibody, at least one receptor, at least one nucleic acid, at least one enzyme, at least one antigen, at least one ligand, at least one adsorbent, or the like, attached thereto. Each second component optionally includes a ligand selected from, e.g., an antigen, a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, an inorganic molecule, an organic molecule, a drug, a receptor ligand, an antibody, a neurotransmitter, a cytokine, a chemokine, a hormone, or the like. Furthermore, each second component typically further includes at least one label (e.g., a fluorescent label, etc.).
In preferred embodiments, the methods typically further include performing a binding assay in which prior to first and second component separation, at least one second component binds to at least one first component, which bound second component subsequently separates from unbound second components. Separation generally results because the bound second component typically has a greater velocity than bulk fluid in the first microchannel, which bulk fluid includes most of the unbound second components. The binding assays optionally include incubating the first and second components together to provide an incubation mixture prior to flowing the particles in the first microchannel. The methods generally also include introducing at least an aliquot of the incubation mixture into the first microchannel by drawing the aliquot from an external source (e.g., a well in a microwell plate or the like) through at least one capillary element that fluidly communicates with the first microchannel. Optionally, after introducing the aliquot of the incubation mixture, the method also includes introducing a selected volume of at least one buffer solution, inter alia, to further effect separation of components flowing in the first microchannel. In certain embodiments, the assays further include incubating a modulator with the first and second components, which modulator modulates the binding of the at least one second component to the at least one first component. For example, in some embodiments, the modulator inhibits the binding of the at least one second component to the at least one first component.
The binding assay generally includes detecting a detectable signal produced by at least the bound second component. For example, in many embodiments, baseline separation of the bound and unbound second components is achieved in less than about 25 seconds. Detecting the detectable signal produced by the separated, bound second component generally increases sensitivity of the binding assay relative to an assay that includes non-separated bound and unbound second components. The detectable signal is optionally selected from, e.g., a refractive index, a cellular activity, a light emission, a change in absorbance, a change in fluorescence, an absorbance, a fluorescence, a color shift, a fluorescence resonance energy transfer, a radioactive emission, a change in pH, a change in temperature, a time resolved fluorescence, a chemiluminescence, a bioluminescence, a change in mass, or the like. Optionally, prior to or after performing the binding assay, the method further includes the step of flowing the first components through the first microchannel to provide a negative control for detecting the detectable signals. As a further option, prior to or after performing the binding assay, the method further includes the step of flowing the second components through the first microchannel to provide a positive control for detecting the detectable signals. As another option, the method further includes quantifying an amount of the bound second component detected, which quantification provides a measure of a concentration of the second component in an incubation mixture that includes the first and second components.
In yet another aspect, the invention also provides particle washing or exchange techniques. For example, focused cells or other particles are optionally washed free of diffusible material by introducing a diluent into the first microchannel from at least a second channel and removing the resulting diluted diffused product comprising diluent mixed with the diffusible material through at least a third microchannel.
Alternating arrangements of diluent input and diffused product output channels are also optionally used to further wash the particles. For example, in one aspect the methods of the invention include simultaneously introducing the diluent into the first microchannel from the second microchannel and a fourth microchannel, where the second and fourth microchannel intersect the first microchannel at a common intersection region. Optionally, the methods include sequentially introducing the diluent into the first microchannel from the second microchannel and a fourth microchannel, wherein the second and fourth microchannels intersect the first microchannel at an offset intersection region. The diffused product is typically removed through the third microchannel and a fifth microchannel, which third and fifth microchannels intersect the first microchannel at a common intersection region. In further washing steps, the diluent is introduced through sixth and seventh microchannels which intersect the first microchannel at a common intersection. The resulting further diluted diffused product is removed through eighth and ninth microchannels, which intersect the first microchannel at a common intersection. Diluent is optionally introduced into the first microchannel by pressure or electrokinetic flow.
In one preferred assay of the invention, the particles are cells and the method includes performing a TUNEL assay or an Annexin-V assay on the cells in the channel to measure apoptosis.
Integrated systems for performing the above methods, including the particle sorting embodiments, are also provided.
An integrated system for providing substantially uniform flow velocity to flowing members of at least one particle population in a microfluidic device optionally includes a body structure that includes at least a first microchannel disposed therein. A cross section of the first microchannel typically includes, e.g., at least one dimension (e.g., a width, a height, or the like) between about 5 xcexcm and about 25 xcexcm or between about 10 xcexcm and about 100 xcexcm. A first fluid direction component (e.g., a fluid pressure force modulator) is typically coupled to the first microchannel for inducing flow of a fluidic material (e.g., a discrete sample volume or the like) that includes the members of the at least one particle population in the first microchannel. The first fluid direction component generally induces non-uniform flow. A source of at least one fluidic material is optionally fluidly coupled to the first microchannel. As an additional option, the source of the at least one fluidic material is fluidly coupled to the first microchannel by at least one capillary element, which capillary element fluidly communicates with the first microchannel. The system also optionally includes at least a second microchannel that intersects the first microchannel for introducing at least one fluid into the first microchannel to horizontally or vertically focus the members of the at least one particle population in the first microchannel. The at least one fluid is optionally introduced using a second fluid direction component that includes one or more of a fluid pressure force modulator, an electrokinetic force modulator, a capillary force modulator, a fluid wicking element, or the like. At least one flow control regulator for regulating flow of the fluidic material or the fluid in the first or second microchannel is also optionally provided. A computer including an instruction set directing simultaneous flow of the members of the at least one particle population in the first microchannel and simultaneous introduction of the at least one fluid from the second microchannel into the first microchannel is optionally also operably coupled to a fluid movement system for directing flow of materials in the microchannels. Typically, the substantially uniform flow velocity increases a rate of particle mixing which reduces dispersion of the members of the at least one particle population in the first microchannel. For example, the members are typically focused proximal to a center portion of the first microchannel, e.g., where focused fluid streamlines, that include the members, flow at higher flow velocities than fluid streamlines flowing proximal to a surface of the first microchannel.
As a further option, this integrated system additionally includes at least a third microchannel which intersects the first microchannel in an intersection region common to the second microchannel. The flow control regulator of this system optionally further regulates flow of the at least one fluid in the second and the third microchannels. In this embodiment, the computer typically also includes an instruction set for simultaneously flowing fluids from the second and third microchannels into the first microchannel.
In particle washing systems, typically, at least fourth and fifth channels which intersect the first microchannel at a common intersection downstream of the second and third microchannels are provided. The computer further includes an instruction set for simultaneously flowing material from the first microchannel into the fourth and fifth microchannels. Sixth and seventh microchannels which intersect the first microchannel at a common intersection downstream of the fourth and fifth microchannels, with the computer further comprising an instruction set for simultaneously flowing material from the sixth and seventh microchannels into the first microchannel are optionally provided. Similarly, eighth and ninth microchannels which intersect the first microchannel at a common intersection downstream of the sixth and seventh microchannels, the computer further including an instruction set for simultaneously flowing material from the first microchannel into the eighth and ninth microchannels are optionally provided.
The integrated system optionally includes sources for any reagent or particle used in the methods noted above, such as one or more sources of terminal deoxynucleotide transferase, one or more sources of one or more fluorescein labeled nucleotides or other labeled polynucleotides, one or more sources of Annexin V, one or more sources of an Annexin V-biotin conjugate, one or more sources of a DNA dye, one or more sources of Campthotecin, one or more sources of Calcein-AM, one or more sources of a control cell, one or more sources of a test cell, etc.
Signal detector(s) mounted proximal to the first microchannel for detecting a detectable signal produced by one or more of the members of the at least one particle population in the microchannel are typically provided in the integrated systems of the invention. The detector also optionally includes, e.g., a fluorescent excitation source and a fluorescent emission detection element. Optionally, the computer is operably linked to the signal detector and has an instruction set for converting detected signal information into digital data.
In certain embodiments of the invention, the system provides substantially uniform flow velocity to members of at least a first of two particle populations. The members of the first particle population typically flow with a greater velocity than bulk fluid in the first microchannel, which bulk fluid generally includes most unbound members of a second particle population. As a result, at least some of the members of the first particle population typically separate from the unbound members of the second particle population. For example, the members of the first particle population typically focus proximal to a center portion of the first microchannel more than the members of the second particle population due to greater dispersion of the members of the second particle population relative to the members of the first particle population in the first microchannel. In particular, the center portion of the first microchannel generally includes a higher velocity flow stream than other flow streams in the first microchannel.
In preferred embodiments, the system is used to perform various binding assays. For example, prior to providing substantially uniform flow velocity, at least one member of the second particle population binds to at least one member of the first particle population, which bound member subsequently separates from the unbound members of the second particle population. Optionally, the members of the first and second particle populations are incubated together to provide an incubation mixture prior to providing the substantially uniform flow velocity. Each member of the first particle population typically includes, e.g., a cell, a bead (e.g., a microbead, etc.) or the like. In addition, each member of the first particle population generally further includes at least one antibody, at least one receptor, at least one nucleic acid, at least one enzyme, at least one antigen, at least one ligand, at least one adsorbent, or the like, attached thereto. Each member of the second particle population optionally includes a ligand selected from, e.g., an antigen, a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, an inorganic molecule, an organic molecule, a drug, a receptor ligand, an antibody, a neurotransmitter, a cytokine, a chemokine, a hormone, or the like. Typically, each member of the second particle population further includes at least one label (e.g., fluorescent label or the like). In these embodiments, the systems typically further includes at least one signal detector mounted proximal to the first microchannel for detecting a detectable signal produced by at least the bound members of the second particle population. For example, the detectable signal is typically selected from, e.g., a refractive index, a cellular activity, a light emission, a change in absorbance, a change in fluorescence, an absorbance, a fluorescence, a color shift, a fluorescence resonance energy transfer, a radioactive emission, a change in pH, a change in temperature, a time resolved fluorescence, a chemiluminescence, a bioluminescence, a change in mass, or the like.
The integrated system of the present invention is also optionally used to sort the members of a particle population (e.g., a cell, a set of cells, a microbead, a set of microbeads, a functionalized microbead, a set of functionalized microbeads, a molecule, a set of molecules, or the like). In this embodiment, the integrated system typically additionally includes a third and a fourth microchannel which intersect the first microchannel downstream from the intersection of the second microchannel with the first microchannel. The fourth microchannel also generally intersects the first microchannel downstream from the intersection of the third microchannel with the first microchannel. The flow control regulator of this system optionally further regulates flow of the at least one fluid in the third or the fourth microchannels. Furthermore, the signal detector typically detects a detectable signal produced by a selected member of the particle population between the intersections of the second and the third microchannels with the first microchannel.
In this particle sorting embodiment, the computer is optionally operably linked to the first or other fluid direction component(s), the flow control regulator, and the signal detector. Additionally, the instruction set typically directs simultaneous introduction of the at least one fluid from the third microchannel into the first microchannel to horizontally or vertically focus the selected member of the particle population such that the selected member is directed into the fourth microchannel in response to the detectable signal produced by the selected member. Optionally, the instruction set further directs simultaneous introduction of the at least one fluid from the third microchannel by activating a heating element (e.g., a Joule heating electrode, a conductively coated microchannel portion, etc.) disposed within the third microchannel or a well that fluidly communicates with the third microchannel.
In another embodiment, at least a portion of the first microchannel optionally includes a separation element disposed therein. The separation element optionally includes, e.g., two sides and at least a portion of the separation element is typically disposed upstream of the fourth microchannel. In this embodiment, a selected member of the particle population is generally directed to one of the two sides of the separation element and into the fourth microchannel that intersects the first microchannel in response to the detectable signal produced by the selected member.
The integrated system for use in particle sorting also optionally includes a fifth microchannel which intersects the first microchannel in an intersection region common to the second microchannel. In this case, the flow control regulator also typically regulates flow of the at least one fluid in the second and the fifth microchannels, and the computer optionally includes an instruction set for simultaneously flowing fluids from the second and the fifth microchannels into the first microchannel. Similarly, the system also optionally includes a sixth microchannel which intersects the first microchannel in an intersection region common to the third microchannel. In this embodiment, the flow control regulator optionally additionally regulates flow of the at least one fluid in the third and the sixth microchannels. Furthermore, the computer also typically includes an instruction set for flowing fluids from the third and the sixth microchannels into the first microchannel. Optionally, the instruction set directs individual or simultaneous fluid flow from the third and sixth microchannels by individually or simultaneously activating at least one heating element (e.g., a Joule heating electrode, a conductively coated microchannel portion, or the like) disposed within each of the third and sixth microchannels or within at least one well that fluidly communicates with each of the third and sixth microchannels.
Many additional aspects of the invention will be apparent upon review, including uses of the devices and systems of the invention, methods of manufacture of the devices and systems of the invention, kits for practicing the methods of the invention and the like. For example, kits comprising any of the devices or systems set forth above, or elements thereof, in conjunction with packaging materials (e.g., containers, sealable plastic bags, etc.) and instructions for using the devices, e.g., to practice the methods herein, are also contemplated.